Production of acid soluble soy protein isolates (“S700”)

ABSTRACT

A soy protein product having a protein content of at least about 60 wt % (N×6.25) d.b., preferably an isolate having a protein content of at least about 90 wt % (N×6.25) d.b., is formed by extracting a soy protein source with a salt solution, preferably aqueous sodium chloride solution, to form an aqueous protein solution having a pH of about 1.5 to 11, preferably about 5 to about 7 and separating the resulting aqueous protein solution from residual soy protein source. The protein concentration of the aqueous protein solution is increased to about 50 to about 400 g/L while the ionic strength is maintained substantially constant by using a selective membrane technique. The resulting concentrated protein solution is optionally diafiltered and a calcium salt, preferably calcium chloride, is added to the concentrated and optionally diafiltered protein solution to a conductivity of 15 to about 85 mS. Precipitate formed as a result of the calcium salt addition is removed and the resulting clarified retentate is diluted into about 2 to about 20 volumes of water prior to acidification to a pH of about 1.5 to about 4.4 to produce an acidified clear protein solution. The acidified clear protein solution is then concentrated and optionally diafiltered and optionally dried. Variations of this procedure can be used to produce a soy protein product which is soluble, transparent and heat stable in acidic aqueous environments.

REFERENCE TO RELATED APPLICATION

This application claims priority under 35 USC 119(e) from U.S.Provisional Patent Application No. 61/213,648 filed Jun. 30, 2009.

FIELD OF INVENTION

The invention relates to the production of soy protein products.

BACKGROUND TO THE INVENTION

In U.S. Provisional Patent Applications Nos. 61/107,112 (7865-373) filedOct. 21, 2008, 61/193,457 (7865-374) filed Dec. 2, 2008, 61/202,070(7865-376) filed Jan. 26, 2009, 60/202,553 filed Mar. 12, 2009(7865-383), 61/213,717 (7865-389) filed Jul. 7, 2009, 61/272,241(7865-400) filed Sep. 3, 2009 and U.S. patent application Ser. No.12/603,087 (7865-415) filed Oct. 21, 2009 (US Patent Publication No.2010-0098818), assigned to the assignee hereof and the disclosures ofwhich are incorporated herein by reference, there is described thepreparation of a soy protein product, preferably a soy protein isolate,which is completely soluble at low pH values and is capable of providingtransparent and heat stable solutions at such low pH values. This soyprotein product may be used for protein fortification of, in particular,soft drinks and sports drinks, as well as other acidic aqueous systems,without precipitation of protein. The soy protein product is produced byextracting a soy protein source with aqueous calcium chloride solutionat natural pH, optionally diluting the resulting aqueous soy proteinsolution, adjusting the pH of the aqueous soy protein solution to a pHof about 1.5 to about 4.4, preferably about 2.0 to about 4.0, to producean acidified clear soy protein solution, which may be optionallyconcentrated and/or diafiltered before drying.

SUMMARY OF THE INVENTION

We have now found that a soy protein product of comparable propertiesmay be formed by a procedure involving extraction of the soy proteinsource with a monovalent salt solution followed by calcium chlorideaddition to the extracted protein solution, before or afterconcentration. Precipitate formed upon addition of the calcium chlorideis removed prior to subsequent processing.

The soy protein product provided herein is soluble at acid pH values toprovide transparent and heat stable aqueous solutions thereof. The soyprotein product may be used for protein fortification of, in particular,soft drinks and sports drinks, without precipitation of protein.

In accordance with one aspect of the present invention, there isprovided a method of producing a soy protein product with a proteincontent of at least about 60 wt % (N×6.25) on a dry weight basis (d.b.),preferably at least about 90 wt %, more preferably at least about 100 wt%, which comprises:

-   -   (a) extracting a soy protein source with a salt solution,        preferably aqueous sodium chloride solution, at a temperature of        at least about 1° C., preferably about 15° to about 35° C., to        cause solubilization of soy protein in the soy protein source        and to form an aqueous protein solution having a protein content        of about 5 to about 50 g/L, preferably about 10 to about 50 g/L,        and a pH of about 1.5 to about 11, preferably about 5 to about        7,    -   (b) separating the aqueous protein solution from residual soy        protein source,    -   (c) increasing the protein concentration of the aqueous protein        solution to about 50 to about 400 g/L, preferably about 100 to        about 250 g/L, while maintaining the ionic strength        substantially constant by using a selective membrane technique        to provide a concentrated protein solution,    -   (d) optionally diafiltering the concentrated protein solution,    -   (e) adding calcium salt solution, preferably aqueous calcium        chloride solution, to the concentrated protein solution to a        conductivity of about 15 to about 85 mS, preferably about 17 to        about 25 mS, to cause a precipitate to form in the concentrated        protein solution,    -   (f) removing the precipitate from the concentrated protein        solution,    -   (g) diluting the clarified concentrated protein solution into        about 2 to about 20 volumes of water, preferably about 10 to        about 15, having a temperature of about 2° to about 90° C.,        preferably about 10° to about 50° C., more preferably about 20°        to about 30° C.,    -   (h) acidifying the resulting solution to a pH of about 1.5 to        about 4.4, preferably about 2.0 to about 4.0, to produce an        acidified clear protein solution,    -   (i) optionally polishing the acidified clear protein solution,    -   (j) increasing the concentration of the acidified clear protein        solution to about 50 to about 300 g/L, preferably about 100 to        about 200 g/L, while maintaining the ionic strength        substantially constant by using a selective membrane technique        to provide a second concentrated protein solution,    -   (k) optionally diafiltering the second concentrated protein        solution, and    -   (l) optionally drying the second concentrated protein solution        to provide a soy protein product having a protein content of at        least about 60 wt % (N×6.25) d.b., preferably at least about 90        wt %, more preferably at least about 100 wt %.

A number of variations of this procedure may be adopted in accordancewith the invention to result in a soy protein product that is soluble,transparent and heat stable in an acidic aqueous environment.

In one such variation, the calcium salt solution, preferably aqueouscalcium chloride, may be added to the aqueous protein solution followingseparation from the soy protein source and prior to concentrating thesolution. Following addition of the calcium chloride, the precipitateformed in the step is removed.

The resulting aqueous soy protein solution may be further processed bythe steps of concentration, dilution, pH adjustment, furtherconcentration and drying, as described above.

Accordingly, in a further aspect of the present invention, there isprovided a method of producing a soy protein product with a proteincontent of at least about 60 wt % (N×6.25) d.b., preferably at leastabout 90 wt %, more preferably at least about 100 wt %, which comprises:

-   -   (a) extracting a soy protein source with a salt solution,        preferably an aqueous sodium chloride solution, at a temperature        of at least about 1° C., preferably about 15° to about 35° C.,        to cause solubilization of soy protein in the soy protein source        and to form an aqueous protein solution having a protein content        of about 5 to about 50 g/L, preferably about 10 to about 50 g/L,        and a pH of about 1.5 to about 11, preferably about 5 to about        7,    -   (b) separating the aqueous protein solution from residual soy        protein source,    -   (c) adding calcium salt solution, preferably aqueous calcium        chloride solution, to the aqueous protein solution to a        conductivity of about 15 to about 85 mS, preferably about 17 to        about 25 mS, to cause a precipitate to form in the aqueous        protein solution,    -   (d) removing the precipitate from the aqueous soy protein        solution,    -   (e) increasing the protein concentration of the soy protein        solution to about 50 to about 400 g/L, preferably about 100 to        about 250 g/L, while maintaining the ionic strength        substantially constant by using a selective membrane technique        to provide a concentrated protein solution,    -   (f) optionally diafiltering the concentrated protein solution,    -   (g) diluting the concentrated and optionally diafiltered protein        solution into about 2 to about 20 volumes of water, preferably        about 10 to about 15, having a temperature of about 2° to about        90° C., preferably about 10° to about 50° C., more preferably        about 20° to about 30° C.,    -   (h) acidifying the resulting solution to a pH of about 1.5 to        about 4.4, preferably about 2.0 to about 4.0, to produce an        acidified clear protein solution,    -   (i) optionally polishing the acidified clear protein solution,    -   (j) increasing the concentration of the acidified clear protein        solution to about 50 to about 300 g/L, preferably about 100 to        about 200 g/L, while maintaining the ionic strength        substantially constant by using a selective membrane technique        to provide a second concentrated protein solution,    -   (k) optionally diafiltering the second concentrated protein        solution, and    -   (l) optionally drying the second concentrated protein solution        to provide a soy protein product having a protein content of at        least about 60 wt % (N×6.25) d.b., preferably at least about 90        wt %, more preferably at least about 100 wt %.

In another variation, the calcium salt solution, preferably aqueouscalcium chloride, may be added to the aqueous protein solution followingseparation from the soy protein source and prior to concentrating thesolution. Following addition of the calcium chloride, the precipitateformed in the step is removed.

The resulting aqueous soy protein solution may be further processed bythe steps of partial concentration, dilution, pH adjustment, furtherconcentration and drying.

Accordingly, in accordance with a further aspect of the presentinvention, there is provided a method of producing a soy protein productwith a protein content of at least about 60 wt % (N×6.25) d.b.,preferably at least about 90 wt %, more preferably at least about 100 wt%, which comprises:

-   -   (a) extracting a soy protein source with a salt solution,        preferably an aqueous sodium chloride solution, at a temperature        of at least about 1° C., preferably about 15° to about 35° C.,        to cause solubilization of soy protein in the soy protein source        and to form an aqueous protein solution having a protein content        of about 5 to about 50 g/L, preferably about 10 to about 50 g/L,        and a pH of about 1.5 to about 11, preferably about 5 to about        7,    -   (b) separating the aqueous protein solution from residual soy        protein source,    -   (c) adding calcium salt solution, preferably aqueous calcium        chloride solution, to the aqueous protein solution to a        conductivity of about 15 to about 85 mS, preferably about 17 to        about 25 mS, to cause a precipitate to form in the aqueous        protein solution,    -   (d) removing the precipitate from the aqueous soy protein        solution,    -   (e) partially concentrating the aqueous protein solution to        about 50 g/L or less while maintaining the ionic strength        substantially constant by using a selective membrane technique        to provide a partially concentrated protein solution,    -   (f) optionally diafiltering the partially concentrated protein        solution,    -   (g) diluting the partially concentrated protein solution into        about 0.5 to about 20 volumes of water, preferably about 1 to        about 10 volumes of water, more preferably about 2 to about 5        volumes of water, having a temperature of about 2° to about 90°        C., preferably about 10° to about 50° C., more preferably about        20° to about 30° C.,    -   (h) acidifying the resulting solution to a pH of about 1.5 to        about 4.4, preferably about 2.0 to about 4.0, to produce an        acidified clear protein solution,    -   (i) optionally polishing the acidified clear protein solution,    -   (j) increasing the concentration of the acidified clear protein        solution to about 50 to about 300 g/L, preferably about 100 to        about 200 g/L, while maintaining the ionic strength        substantially constant by using a selective membrane technique        to provide a concentrated protein solution,    -   (k) optionally diafiltering the concentrated protein solution,        and    -   (l) optionally drying the concentrated protein solution to        provide a soy protein product having a protein content of at        least about 60 wt % (N×6.25) d.b., preferably at least about 90        wt %, more preferably at least about 100 wt %.

In another variation, the calcium salt solution, preferably aqueouscalcium chloride may be added to the aqueous soy protein solutionfollowing separation from the soy protein source and prior toconcentrating the solution. Following addition of the calcium chloride,the precipitate formed in the step is removed.

The resulting aqueous soy protein solution may be diluted to decreasethe conductivity, such as by one volume of water, and then adjusted inpH with acid. The acidified solution may then be concentrated anddiafiltered to further decrease the conductivity resulting in a clear,low pH solution ready for drying.

Accordingly, in another aspect of the present invention, there isprovided a method of producing a soy protein product with a proteincontent of at least about 60 wt % (N×6.25) d.b., preferably at leastabout 90 wt %, more preferably at least about 100 wt %, which comprises:

-   -   (a) extracting a soy protein source with a salt solution,        preferably an aqueous sodium chloride solution, at a temperature        of at least about 1° C., preferably about 15° to about 35° C.,        to cause solubilization of soy protein in the soy protein source        and to form an aqueous protein solution having a protein content        of about 5 to about 50 g/L, preferably about 10 to about 50 g/L,        and a pH of about 1.5 to about 11, preferably about 5 to about        7,    -   (b) separating the aqueous protein solution from residual soy        protein source,    -   (c) adding calcium salt solution, preferably aqueous calcium        chloride solution, to the aqueous protein solution to a        conductivity of about 15 to about 85 mS, preferably about 17 to        about 25 mS, to cause a precipitate to form in the aqueous        protein solution,    -   (d) removing the precipitate from the protein solution,    -   (e) diluting the clarified solution with about 0.5 to about 10        volumes of water, preferably about 0.5 to about 2 volumes of        water, having a temperature of about 2° C. to about 90° C.,        preferably about 10° C. to about 50° C., more preferably about        20° C. to about 30° C.    -   (f) acidifying the resulting solution to a pH of about 1.5 to        about 4.4, preferably about 2.0 to about 4.0, to produce an        acidified clear protein solution,    -   (g) optionally polishing the acidified clear protein solution,    -   (h) increasing the concentration of the acidified clear protein        solution to a protein concentration of about 50 to about 300        g/L, preferably about 100 to about 200 g/L, while maintaining        the ionic strength substantially constant by using a selective        membrane technique to provide a concentrated protein solution,    -   (i) optionally diafiltering the concentrated protein solution,        and    -   (j) optionally drying the concentrated protein solution to        provide a soy protein product having a protein content of at        least about 60 wt % (N×6.25) d.b., preferably at least about 90        wt %, more preferably at least about 100 wt %.

In another such variation, the calcium salt solution, preferably aqueouscalcium chloride may be added to partially concentrated soy proteinsolution and the resulting precipitate removed from the partiallyconcentrated soy protein solution. The clarified solution may then beput back on the membrane system for additional concentration prior tothe dilution, pH adjustment, further concentrating and drying stepsdescribed above.

Accordingly, in an additional aspect of the invention, there is provideda method of producing a soy protein product with a protein content of atleast about 60 wt % (N×6.25), preferably at least about 90 wt %, morepreferably at least about 100 wt %, which comprises:

-   -   (a) extracting a soy protein source with a salt solution,        preferably aqueous sodium chloride solution, at a temperature of        at least about 1° C., preferably about 15° to about 35° C., to        cause solubilization of soy protein in the soy protein source        and to form an aqueous protein solution having a protein content        of about 5 to about 50 g/L, preferably about 10 to about 50 g/L,        and a pH of about 1.5 to about 11, preferably about 5 to about        7,    -   (b) separating the aqueous protein solution from residual soy        protein source,    -   (c) partially concentrating the aqueous protein solution to        about 50 g/L or less while maintaining the ionic strength        substantially constant by using a selective membrane technique        to provide a partially concentrated protein solution,    -   (d) optionally diafiltering the partially concentrated protein        solution,    -   (e) adding calcium salt solution to the partially concentrated        protein solution to a conductivity of about 15 mS to about 85        mS, preferably about 17 mS to about 25 mS, to cause a        precipitate to form in the partially concentrated protein        solution,    -   (f) removing the precipitate from the partially concentrated        protein solution,    -   (g) further increasing the protein concentration of the        partially concentrated protein solution to about 50 to about 400        g/L, preferably about 100 to about 250 g/L, while maintaining        the ionic strength substantially constant by using a selective        membrane technique to provide a concentrated protein solution,    -   (h) optionally diafiltering the concentrated protein solution,    -   (i) diluting the concentrated protein solution into about 2 to        about 20 volumes of water, preferably about 10 to about 15,        having a temperature of about 2° to about 90° C., preferably        about 10° to about 50° C., more preferably about 20° to about        30° C.,    -   (j) acidifying the resulting solution to a pH of about 1.5 to        about 4.4, preferably about 2.0 to about 4.0, to produce an        acidified clear protein solution,    -   (k) optionally polishing the acidified clear protein solution,    -   (j) increasing the concentration of the acidified clear protein        solution to a protein concentration of about 50 to about 300        g/L, preferably about 100 to about 200 g/L, while maintaining        the ionic strength substantially constant by using a selective        membrane technique to provide a second concentrated protein        solution,    -   (k) optionally diafiltering the second concentrated protein        solution, and    -   (l) optionally drying the second concentrated protein solution        to provide a soy protein product having a protein content of at        least about 60 wt % (N×6.25) d.b., preferably at least about 90        wt %, more preferably at least about 100 wt %.

Alternatively, the partially concentrated soy protein solution,clarified after calcium chloride treatment, may be diluted sufficientlyto decrease the conductivity, pH adjusted and then concentrated anddiafiltered prior to drying.

Accordingly, in a further aspect of the invention, there is provided amethod of producing a soy protein product with a protein content of atleast about 60 wt % (N×6.25), preferably at least about 90 wt %, morepreferably at least about 100 wt %, which comprises:

-   -   (a) extracting a soy protein source with a salt solution,        preferably an aqueous sodium chloride solution, at a temperature        of at least about 1° C., preferably about 15° to about 35° C.,        to cause solubilization of soy protein in the soy protein source        and to form an aqueous protein solution having a protein content        of about 5 to about 50 g/L, preferably about 10 to about 50 g/L,        and a pH of about 1.5 to about 11, preferably about 5 to about        7,    -   (b) separating the aqueous protein solution from residual soy        protein source,    -   (c) partially concentrating the aqueous protein solution to a        protein concentration of about 50 g/L or less while maintaining        the ionic strength substantially constant by using a selective        membrane technique to provide a partially concentrated protein        solution,    -   (d) optionally diafiltering the partially concentrated protein        solution,    -   (e) adding calcium salt solution, preferably aqueous calcium        chloride solution, to the partially concentrated protein        solution to a conductivity of 15 mS to about 85 mS, preferably        about 17 mS to about 25 mS, to cause a precipitate to form in        the partially concentrated protein solution,    -   (f) removing the precipitate from the partially concentrated        protein solution,    -   (g) diluting the clarified partially concentrated protein        solution into about 0.5 to about 20 volumes of water, preferably        about 1 to about 10 volumes of water, more preferably about 2 to        about 5 volumes of water, having a temperature of about 2° to        about 90° C., preferably about 10° to about 50° C., more        preferably about 20° to about 30° C.,    -   (h) acidifying the resulting solution to a pH of about 1.5 to        about 4.4, preferably about 2.0 to about 4.0, to produce an        acidified clear protein solution,    -   (i) optionally polishing the acidified clear protein solution,    -   (j) increasing the concentration of the acidified clear protein        solution to about 50 to about 300 g/L, preferably about 100 to        about 200 g/L, while maintaining the ionic strength        substantially constant by using a selective membrane technique        to provide a concentrated protein solution,    -   (k) optionally diafiltering the concentrated protein solution,        and    -   (l) optionally drying the concentrated protein solution to        provide a soy protein product having a protein content of at        least about 60 wt % (N×6.25) d.b., preferably at least about 90        wt %, more preferably at least about 100 wt %.

While the present invention refers mainly to the production of soyprotein isolates, it is contemplated that soy protein products of lesserpurity may be provided with substantially similar properties to the soyprotein isolates. Such lesser purity products may have a proteinconcentration of at least about 60% by weight (N×6.25) d.b.

The novel soy protein product of the invention can be blended withpowdered drinks for the formation of aqueous soft drinks or sportsdrinks by dissolving the same in water. Such blend may be a powderedbeverage.

The soy protein product provided herein may be provided as an aqueoussolution thereof having a high degree of clarity at acid pH values andwhich is heat stable at these pH values.

In another aspect of the present invention, there is provided an aqueoussolution of the soy product provided herein which is heat stable at lowpH. The aqueous solution may be a beverage, which may be a clearbeverage in which the soy protein product is completely soluble andtransparent or an opaque beverage in which the soy protein product doesnot increase the opacity. The soy protein product also has goodsolubility at about pH 7.5 to about pH 8.0, providing aqueous solutionswith good clarity and heat stability. An aqueous solution of the soyprotein product prepared at about pH 7.5 to about pH 8 may be abeverage.

The soy protein products produced according to the process herein lackthe characteristic beany flavour of soy protein isolates and aresuitable, not only for protein fortification of acidic media, but may beused in a wide variety of conventional applications of protein isolates,including but not limited to protein fortification of processed foodsand beverages, emulsification of oils, as a body former in baked goodsand foaming agent in products which entrap gases. In addition, the soyprotein product may be formed into protein fibers, useful in meatanalogs, may be used as an egg white substitute or extender in foodproducts where egg white is used as a binder. The soy protein productmay be used in nutritional supplements. Other uses of the soy proteinproducts are in pet foods, animal feed and in industrial and cosmeticapplications and in personal care products.

GENERAL DESCRIPTION OF INVENTION

The initial step of the process of providing the soy protein productinvolves solubilizing soy protein from a soy protein source. The soyprotein source may be soybeans or any soy product or by-product derivedfrom the processing of soybeans including but not limited to soy meal,soy flakes, soy grits and soy flour. The soy protein source may be usedin the full fat form, partially defatted form or fully defatted form.Where the soy protein source contains an appreciable amount of fat, anoil-removal step generally is required during the process. The soyprotein recovered from the soy protein source may be the proteinnaturally occurring in soybean or the proteinaceous material may be aprotein modified by genetic manipulation but possessing characteristichydrophobic and polar properties of the natural protein.

Protein solubilization may be effected by using a food grade saltsolution such as a solution of food grade sodium chloride. Where the soyprotein product is intended for non-food uses, non-food-grade chemicalsmay be used. Other monovalent salts also may be used, such as potassiumchloride. As the concentration of the salt solution increases, thedegree of solubilization of protein from the soy protein sourceinitially increases until a maximum value is achieved. Any subsequentincrease in salt concentration does not increase the total proteinsolubilized. The concentration of the salt solution which causes maximumprotein solubilization varies depending on the salt concerned. It isusually preferred to utilize a concentration value less than about 1.0M, and more preferably a value of about 0.10 M to about 0.15 M.

In a batch process, the salt solubilization of the protein is effectedat a temperature of from about 1° C. to about 100° C., preferably about15° to about 35° C., preferably accompanied by agitation to decrease thesolubilization time, which is usually about 1 to about 60 minutes. It ispreferred to effect the solubilization to extract substantially as muchprotein from the soy protein source as is practicable, so as to providean overall high product yield.

In a continuous process, the extraction of the protein from the soyprotein source is carried out in any manner consistent with effecting acontinuous extraction of protein from the soy protein source. In oneembodiment, the soy protein source is continuously mixed with a foodgrade salt solution and the mixture is conveyed through a pipe orconduit having a length and at a flow rate for a residence timesufficient to effect the desired extraction in accordance with theparameters described herein. In such continuous procedure, the proteinsolubilization step is effected rapidly, in a time of up to about 10minutes, preferably to effect solubilization to extract substantially asmuch protein from the soy protein source as is practicable. Thesolubilization in the continuous procedure is effected at temperaturesbetween about 1° C. and about 100° C., preferably between about 15° C.and about 35° C.

The extraction may be carried out at the natural pH of the soy proteinsource/salt solution system, generally about 5 to about 7.Alternatively, the pH of the extraction may be adjusted to any desiredvalue within the range of about 1.5 to about 11, preferably about 5 toabout 7, by the use of any convenient acid, usually hydrochloric acid,or alkali, usually sodium hydroxide, as required.

The concentration of soy protein source in the food grade salt solutionduring the solubilization step may vary widely. Typical concentrationvalues are about 5 to about 15% w/v.

The protein extraction step with the aqueous salt solution has theadditional effect of solubilizing fats which may be present in the soyprotein source, which then results in the fats being present in theaqueous phase.

The protein solution resulting from the extraction step generally has aprotein concentration of about 5 to about 50 g/L, preferably about 10 toabout 50 g/L.

The aqueous salt solution may contain an antioxidant. The antioxidantmay be any convenient antioxidant, such as sodium sulfite or ascorbicacid. The quantity of antioxidant employed may vary from about 0.01 toabout 1 wt % of the solution, preferably about 0.05 wt %. Theantioxidant serves to inhibit the oxidation of any phenolics in theprotein solution.

The aqueous phase resulting from the extraction step then may beseparated from the residual soy protein source, in any convenientmanner, such as by employing a decanter centrifuge, followed by disccentrifugation and/or filtration to remove residual soy protein sourcematerial. The separated residual soy protein source may be dried fordisposal. Alternatively, the separated residual soy protein source maybe processed to recover some residual protein. For example, theseparated residual soy protein source may be processed by a conventionalisoelectric precipitation procedure or any other convenient procedure torecover such residual protein.

Where the soy protein source contains significant quantities of fat, asdescribed in U.S. Pat. Nos. 5,844,086 and 6,005,076, assigned to theassignee hereof and the disclosures of which are incorporated herein byreference, then the defatting steps described therein may be effected onthe separated aqueous protein solution. Alternatively, defatting of theaqueous protein solution may be achieved by any other convenientprocedure.

The aqueous soy protein solution may be treated with an adsorbent, suchas powdered activated carbon or granulated activated carbon, to removecolour and/or odour compounds. Such adsorbent treatment may be carriedout under any convenient conditions, generally at the ambienttemperature of the separated aqueous protein solution. For powderedactivated carbon, an amount of about 0.025% to about 5% w/v, preferablyabout 0.05% to about 2% w/v, is employed. The adsorbing agent may beremoved from the soy protein solution by any convenient means, such asby filtration.

As an alternative to extracting the soy protein source with an aqueoussalt solution, such extraction may be made using water alone. Where suchalternative is employed, then the salt, in the concentrations discussedabove may be added to the protein solution after separation from theresidual soy protein source. When a first fat removal step is carriedout, the salt is generally added after completion of such operations.

As an alternative to processing the aqueous protein solution at the pHof extraction, the aqueous soy protein solution resulting from theextraction step may be pH adjusted to the range of about 5 to about 7,prior to further processing as discussed below. Such pH adjustment maybe effected using any convenient acid, such as hydrochloric acid, oralkali, such as sodium hydroxide, as appropriate. If necessary, theprotein solution may be clarified by any convenient procedure such ascentrifugation or filtration after the pH adjustment and prior tofurther processing.

The aqueous soy protein solution is then concentrated to increase theprotein concentration thereof while maintaining the ionic strengththereof substantially constant. Such concentration generally is effectedto provide a concentrated protein solution having a proteinconcentration of about 50 to about 400 g/L, preferably about 100 toabout 250 g/L.

The concentration step may be effected in any convenient mannerconsistent with batch or continuous operation, such as by employing anyconvenient selective membrane technique, such as ultrafiltration ordiafiltration, using membranes, such as hollow-fibre membranes orspiral-wound membranes, with a suitable molecular weight cut-off, suchas about 3,000 to about 1,000,000 Daltons, preferably about 5,000 toabout 100,000 Daltons, having regard to differing membrane materials andconfigurations, and, for continuous operation, dimensioned to permit thedesired degree of concentration as the aqueous protein solution passesthrough the membranes.

As is well known, ultrafiltration and similar selective membranetechniques permit low molecular weight species to pass therethroughwhile preventing higher molecular weight species from so doing. The lowmolecular weight species include not only the ionic species of the foodgrade salt but also low molecular weight materials extracted from thesource material, such as carbohydrates, pigments, low molecular weightproteins and anti-nutritional factors, such as trypsin inhibitors, whichthemselves are low molecular weight proteins. The molecular weightcut-off of the membrane is usually chosen to ensure retention of asignificant proportion of the protein in the solution, while permittingcontaminants to pass through having regard to the different membranematerials and configurations.

The protein solution may be subjected to a diafiltration step, before orafter complete concentration, preferably using an aqueous salt solutionof the same molarity and pH as the extraction solution. Suchdiafiltration may be effected using from about 2 to about 40 volumes ofdiafiltration solution, preferably about 5 to about 25 volumes ofdiafiltration solution. In the diafiltration operation, furtherquantities of contaminants are removed from the aqueous soy proteinsolution by passage through the membrane with the permeate. Thediafiltration operation may be effected until no significant furtherquantities of contaminants or visible colour are present in thepermeate. Such diafiltration may be effected using the same membrane asfor the concentration step. However, if desired, the diafiltration stepmay be effected using a separate membrane with a different molecularweight cut-off, such as a membrane having a molecular weight cut-off inthe range of about 3,000 to about 1,000,000 Daltons, preferably about5,000 to about 100,000 Daltons, having regard to different membranematerials and configuration.

An antioxidant may be present in the diafiltration medium during atleast part of the diafiltration step. The antioxidant may be anyconvenient antioxidant, such as sodium sulfite or ascorbic acid. Thequantity of antioxidant employed in the diafiltration medium depends onthe materials employed and may vary from about 0.01 to about 1 wt %,preferably about 0.05 wt %. The antioxidant serves to inhibit theoxidation of any phenolics present in the soy protein solution.

The concentration step and the diafiltration step may be effected at anyconvenient temperature, generally about 2° to about 60° C., preferablyabout 20° to about 35° C., and for the period of time to effect thedesired degree of concentration and diafiltration. The temperature andother conditions used to some degree depend upon the membrane equipmentused to effect the membrane processing, the desired proteinconcentration of the solution and the efficiency of removal ofcontaminants to the permeate.

For example, the concentration and/or diafiltration steps may beoperated in a manner favorable for removal of trypsin inhibitors in thepermeate along with the other contaminants. Removal of the trypsininhibitors is promoted by using a membrane of larger pore size, such asabout 30,000 to about 1,000,000 Da, operating the membrane at elevatedtemperatures, such as about 30 to about 60° C. and employing greatervolumes of diafiltration medium, such as about 20 to about 40 volumes.

Further, a reduction in trypsin inhibitor activity may be achieved byexposing soy materials to reducing agents that disrupt or rearrange thedisulfide bonds of the inhibitors. Suitable reducing agents includesodium sulfite, cysteine and N-acetylcysteine.

The addition of such reducing agents may be effected at various stagesof the overall process. The reducing agent may be added with the soyprotein source material in the extraction step, may be added to theclarified aqueous soy protein solution following removal of residual soyprotein source material, may be added to the concentrated proteinsolution before or after diafiltration, may be added to the acidified,concentrated protein solution before or after diafiltration, or may bedry blended with the dried soy protein product. The addition of thereducing agent may be combined with the membrane processing steps, asdescribed above or a heat treatment step as described below.

If it is desired to retain active trypsin inhibitors in the concentratedprotein solution, this can be achieved by utilizing a concentration anddiafiltration membrane with a smaller pore size, operating the membraneat lower temperatures, employing fewer volumes of diafiltration mediumand not employing a reducing agent.

The concentrated and optionally diafiltered protein solution may besubject to a further defatting operation, if required, as described inU.S. Pat. Nos. 5,844,086 and 6,005,076. Alternatively, defatting of theconcentrated and optionally diafiltered protein solution may be achievedby any other convenient procedure.

The concentrated and optionally diafiltered aqueous protein solution maybe treated with an adsorbent, such as powdered activated carbon orgranulated activated carbon, to remove colour and/or odour compounds.Such adsorbent treatment may be carried out under any convenientconditions, generally at the ambient temperature of the concentratedprotein solution. For powdered activated carbon, an amount of about0.025% to about 5% w/v, preferably about 0.05% to about 2% w/v, isemployed. The adsorbent may be removed from the soy protein solution byany convenient means, such as by filtration.

The concentrated and optionally diafiltered soy protein solution may besubjected to pasteurization to reduce the microbial load. Suchpasteurization may be effected under any desired pasteurizationconditions. Generally, the concentrated and optionally diafiltered soyprotein solution is heated to a temperature of about 55° to about 70°C., preferably about 60° to about 65° C., for about 30 seconds to about60 minutes, preferably about 10 minutes to about 15 minutes. Thepasteurized concentrated soy protein solution then may be cooled forfurther processing as described below, preferably to a temperature ofabout 20° to about 35° C.

Following the concentration step and optional diafiltration, defatting,adsorbent treatment and pasteurization steps, a calcium salt, usuallycalcium chloride solution, is added to the resulting solution. Thisaddition causes the formation of a precipitate containing primarilyphytate. Sufficient calcium chloride is added to provide a solutionhaving a conductivity generally of about 15 to about 85 mS, preferablyof about 17 to about 25 mS.

Although the addition of calcium salt is usually effected using calciumchloride solution, solutions of other calcium salts may be used.Alternatively, the calcium salt may be added in a dry form. In addition,other alkaline earth metal salts may be used.

The addition of the calcium salt may be effected at a temperature ofabout 2° to about 70° C., preferably about 20° C. to about 35° C.Following addition of the calcium salt, the precipitated material isremoved from the protein solution by any convenient means, such as bycentrifugation or filtration.

The concentrated protein solution from the phytate precipitation is thendiluted by mixing the retentate with water having a volume required toachieve the degree of dilution desired. The concentrated proteinsolution generally is diluted about 2 to about 20 fold, preferably about10 to about 15 fold. The water with which the concentrated proteinsolution is mixed has a temperature of about 2° to about 90° C.,preferably about 10° to about 50° C., more preferably about 20° to about30° C. Dilution of the concentrated protein solution results in theformation of a protein precipitate. Acidification of the dilutedsolution resolubilizes the protein and results in a transparent solutionfurther processed as detailed below. Alternatively, the precipitate maybe collected and dried by any convenient means.

The diluted retentate then is adjusted in pH to about 1.5 to about 4.4,preferably about 2.0 to about 4.0, by the addition of any suitable acid,such as hydrochloric acid or phosphoric acid, to result in a clearaqueous soy protein solution. The diluted and acidified protein solutionmay optionally be polished by any convenient means such as filtration.

The acidified clear soy protein solution may be subjected to a heattreatment to inactivate heat labile anti-nutritional factors, such astrypsin inhibitors, present in such solution as a result of extractionfrom a soy protein source material during the extraction step. Such aheating step also provides the additional benefit of reducing themicrobial load. Generally, the protein solution is heated to atemperature of about 70° to about 160° C., preferably about 80° to about120° C., more preferably about 85° C. to about 95° C., for about 10seconds to about 60 minutes, preferably about 30 seconds to about 5minutes. The heat treated acidified soy protein solution then may becooled for further processing as described below, preferably to atemperature of about 2° C. to about 60° C., more preferably about 20° C.to about 35° C.

The acidified clear soy protein solution is concentrated to increase theprotein concentration thereof while maintaining the ionic strengththereof substantially constant. Such concentration generally is effectedto provide a concentrated protein solution having a proteinconcentration of about 50 to about 300 g/L, preferably about 100 toabout 200 g/L.

The concentration step may be effected in any convenient mannerconsistent with batch or continuous operation, such as by employing anyconvenient selective membrane technique, such as ultrafiltration ordiafiltration, using membranes, such as hollow-fibre membranes orspiral-wound membranes, with a suitable molecular weight cut-off, suchas about 3,000 to about 1,000,000 Daltons, preferably about 5,000 toabout 100,000 Daltons, having regard to differing membrane materials andconfigurations, and, for continuous operation, dimensioned to permit thedesired degree of concentration as the aqueous protein solution passesthrough the membranes.

As is well known, ultrafiltration and similar selective membranetechniques permit low molecular weight species to pass therethroughwhile preventing higher molecular weight species from so doing. The lowmolecular weight species include not only the ionic species of the foodgrade salt but also low molecular weight materials extracted from thesource material, such as, carbohydrates, pigments, low molecular weightproteins and anti-nutritional factors. The molecular weight cut-off ofthe membrane is usually chosen to ensure retention of a significantproportion of the protein in the solution, while permitting contaminantsto pass through having regard to the different membrane materials andconfigurations.

The protein solution may be subjected to a diafiltration step, before orafter complete concentration, using water or a dilute saline solution.The diafiltration solution may be at its natural pH or at a pH equal tothe protein solution being diafiltered or at any pH value in between.Such diafiltration may be effected using from about 2 to about 40volumes of diafiltration solution, preferably about 5 to about 25volumes of diafiltration solution. In the diafiltration operation,further quantities of contaminants are removed from the clear aqueoussoy protein solution by passage through the membrane with the permeate.The diafiltration operation may be effected until no significant furtherquantities of contaminants or visible colour are present in the permeateor until the retentate has been sufficiently purified so as, when dried,to provide a soy protein isolate with a protein content of at leastabout 90 wt % (N×6.25) d.b. Such diafiltration may be effected using thesame membrane as for the concentration step. However, if desired, thediafiltration step may be effected using a separate membrane with adifferent molecular weight cut-off, such as a membrane having amolecular weight cut-off in the range of about 3,000 to about 1,000,000Daltons, preferably about 5,000 to about 100,000 Daltons, having regardto different membrane materials and configuration.

An antioxidant may be present in the diafiltration medium during atleast part of the diafiltration step. The antioxidant may be anyconvenient antioxidant, such as sodium sulfite or ascorbic acid. Thequantity of antioxidant employed in the diafiltration medium depends onthe materials employed and may vary from about 0.01 to about 1 wt %,preferably about 0.05 wt %. The antioxidant serves to inhibit theoxidation of any phenolics present in the soy protein solution.

The concentration step and the optional diafiltration step may beeffected at any convenient temperature, generally about 2° C. to about60° C., preferably about 20° C. to about 35° C., and for the period oftime to effect the desired degree of concentration and diafiltration.The temperature and other conditions used to some degree depend upon themembrane equipment used to effect the membrane processing, the desiredprotein concentration of the solution and the efficiency of the removalof contaminants to the permeate.

There are two main trypsin inhibitors in soy, namely the Kunitzinhibitor, which is a heat-labile molecule with a molecular weight ofapproximately 21,000 Daltons, and the Bowman-Birk inhibitor, a moreheat-stable molecule with a molecular weight of about 8,000 Daltons. Thelevel of trypsin inhibitor activity in the final soy protein product canbe controlled by manipulation of various process variables.

As noted above, heat treatment of the acidified clear soy proteinsolution may be used to inactivate heat-labile trypsin inhibitors. Thepartially concentrated or fully concentrated acidified clear soy proteinsolution may also be heat treated to inactivate heat labile trypsininhibitors.

Acidifying and membrane processing the diluted protein solution at alower pH (1.5 to 3.0) may reduce the trypsin inhibitor activity relativeto processing the solution at higher pH (3.0 to 4.4). When the proteinsolution is concentrated and diafiltered at the low end of the pH range,it may be desired to raise the pH of the retentate prior to drying. ThepH of the concentrated and diafiltered protein solution may be raised tothe desired value, for example pH 3, by the addition of any convenientfood grade alkali such as sodium hydroxide.

As mentioned above, the concentration and/or diafiltration steps may beoperated in a manner favorable for removal of trypsin inhibitors in thepermeate along with the other contaminants. Removal of the trypsininhibitors is promoted by using a membrane of larger pore size, such asabout 30,000 to about 1,000,000 Da, operating the membrane at elevatedtemperatures, such as about 30 to about 60° C. and employing greatervolumes of diafiltration medium, such as about 20 to about 40 volumes.

Further, a reduction in trypsin inhibitor activity may be achieved byexposing soy materials to reducing agents that disrupt or rearrange thedisulfide bonds of the inhibitors. Suitable reducing agents includesodium sulfite, cysteine and N-acetylcysteine.

If it is desired to retain active trypsin inhibitors in the concentratedprotein solution, this can be achieved by utilizing a concentration anddiafiltration membrane with a smaller pore size, operating the membraneat lower temperatures, employing fewer volumes of diafiltration mediumand not employing a reducing agent.

The concentrated and optionally diafiltered aqueous acidified proteinsolution may be treated with an adsorbent, such as powdered activatedcarbon or granulated activated carbon, to remove colour and/or odourcompounds. Such adsorbent treatment may be carried out under anyconvenient conditions, generally at the ambient temperature of theconcentrated protein solution. For powdered activated carbon, an amountof about 0.025% to about 5% w/v, preferably about 0.05% to about 2% w/v,is employed. The adsorbent may be removed from the soy protein solutionby any convenient means, such as by filtration.

The concentrated and optionally diafiltered acidified clear aqueous soyprotein solution may be dried by any convenient technique, such as spraydrying or freeze drying. The pasteurization step described above may beeffected on the soy protein solution prior to drying. The dry soyprotein product has a protein content, in excess of about 60 wt %protein. Preferably the dry soy protein product is an isolate containingat least about 90 wt % protein, more preferably at least about 100 wt %(N×6.25). The soy protein product is low in phytic acid content,generally less than about 1.5% by weight. By partially concentratingand/or partially diafiltering the aqueous soy protein solution, it ispossible to only partially remove contaminants and thereby result in adry soy protein product of lesser purity.

As noted above there are several variations on the procedure describedherein to produce the soy protein product and involve severalmodifications to the steps outlined herein.

The soy protein product produced herein is soluble in an acidic aqueousenvironment, making the product ideal for incorporation into beverages,both carbonated and uncarbonated, to provide protein fortificationthereto. Such beverages have a wide range of acidic pH values, rangingfrom about 2.5 to about 5. The soy protein product provided herein maybe added to such beverages in any convenient quantity to provide proteinfortification to such beverages, for example, to supply at least about 5g of soy protein per serving. The added soy protein product dissolves inthe beverage and does not impair the clarity of the beverage, even afterthermal processing. The soy protein product may be blended with driedbeverage prior to reconstitution of the beverage by dissolution inwater. In some cases, modification of the normal formulation may benecessary where components present in the beverage may adversely affectthe ability of the composition to remain dissolved in the beverage.

EXAMPLES Example 1

This Example illustrates the preparation of a dried soy protein isolatein accordance with one embodiment of the invention.

20 kg of defatted, minimally heat treated soy flour was added to 200 Lof 0.15M sodium chloride solution at ambient temperature and agitatedfor 30 minutes to provide an aqueous protein solution. The bulk of theresidual soy flour was removed by centrifugation to provide 165.4 L ofsolution having a protein content of 2.14% by weight. Sufficient calciumchloride was added to raise the conductivity of the solution to 22 mSand a precipitate formed. This precipitate was removed by centrifugationto provide 156.2 L of solution having a protein content that was notdetermined. This solution was combined with 156.2 L of reverse osmosispurified water and the pH lowered to 3 by the addition of diluted HCl.The resulting solution had a protein content of 0.65% by weight and aconductivity of 13.37 mS. This solution was polished by filtration. Postclarification, a total volume of 350 L of solution was obtained having aprotein content of 0.53% by weight.

The 350 L of filtered protein solution was reduced in volume to 26.42 kgby concentration on a PVDF membrane having a molecular weight cutoff of5,000 daltons. The concentrated protein solution was then diafilteredwith 125 L of reverse osmosis purified water that had been adjusted topH 3 with diluted HCl. The resulting diafiltered, concentrated proteinsolution had a protein content of 6.99% by weight and represented ayield of 72.6 wt % of the initial filtered protein solution. Thediafiltered, concentrated protein solution was then dried to yield aproduct found to have a protein content of 101.44% (N×6.25) d.b. Theproduct was termed S005-A19-09A S700.

A 3.2% w/v protein solution of S005-A19-09A S700 was prepared in waterand the colour and clarity assessed using a HunterLab ColorQuest XEinstrument operated in transmission mode. The pH of the solution wasmeasured with a pH meter.

The pH, colour and clarity values are set forth in the following Table1:

TABLE 1 pH and HunterLab scores for 3.2% protein solution ofS005-A19-09A S700 Sample pH L* a* b* Haze (%) S005-A19-09A S700 3.0296.95 −0.87 8.72 2.3

As may be seen from Table 1, the colour of the S700 solution was verylight and very little haze was detected.

The colour of the dry powder was also assessed with the HunterLabColorQuest XE instrument in reflectance mode. The colour values are setforth in the following Table 2:

TABLE 2 HunterLab scores for S005-A19-09A S700 dry powder Sample L* a*b* S005-A19-09A S700 87.32 −0.04 9.59

As may be seen from Table 2, the dry product was very light in colour.

Example 2

This Example contains an evaluation of the heat stability in water ofthe soy protein isolate produced by the method of Example 1 (S700).

A 2% w/v protein solution of S005-A19-09A S700 in water was produced andthe pH adjusted to 3. The clarity of this solution was assessed by hazemeasurement with the HunterLab ColorQuest XE instrument. The solutionwas then heated to 95° C., held at this temperature for 30 seconds andthen immediately cooled to room temperature in an ice bath. The clarityof the heat treated solution was then measured again.

The clarity of the protein solution before and after heating is setforth in the following Table 3:

TABLE 3 Effective of heat treatment on clarity of S005-A19-09A S700solution Sample Haze (%) Before heating 1.4 After heating 2.5

As can be seen from the results in Table 3, the initial solution of S700had very little haze, as did the heat-treated sample.

Example 3

This Example contains an evaluation of the solubility in water of thesoy protein isolate produced by the method of Example 1 (S700).Solubility was tested based on protein solubility (termed proteinmethod, a modified version of the procedure of Morr et al., J. Food Sci.50:1715-1718) and total product solubility (termed pellet method).

Sufficient protein powder to supply 0.5 g of protein was weighed into abeaker and then a small amount of reverse osmosis (RO) purified waterwas added and the mixture stirred until a smooth paste formed.Additional water was then added to bring the volume to approximately 45ml. The contents of the beaker were then slowly stirred for 60 minutesusing a magnetic stirrer. The pH was determined immediately afterdispersing the protein and was adjusted to the appropriate level (2, 3,4, 5, 6 or 7) with diluted NaOH or HCl. A sample was also prepared atnatural pH. For the pH adjusted samples, the pH was measured andcorrected two times during the 60 minutes stirring. After the 60 minutesof stirring, the samples were made up to 50 ml total volume with ROwater, yielding a 1% w/v protein dispersion. The protein content of thedispersions was measured using a Leco FP528 Nitrogen Determinator.Aliquots (20 ml) of the dispersions were then transferred to pre-weighedcentrifuge tubes that had been dried overnight in a 100° C. oven thencooled in a desiccator and the tubes capped. The samples werecentrifuged at 7800 g for 10 minutes, which sedimented insolublematerial and yielded a clear supernatant. The protein content of thesupernatant was measured by Leco analysis and then the supernatant andthe tube lids were discarded and the pellet material dried overnight inan oven set at 100° C. The next morning the tubes were transferred to adesiccator and allowed to cool. The weight of dry pellet material wasrecorded. The dry weight of the initial protein powder was calculated bymultiplying the weight of powder used by a factor of ((100−moisturecontent of the powder (%))/100). Solubility of the product was thencalculated two different ways:Solubility (protein method) (%)=(% protein in supernatant/% protein ininitial dispersion)×100  1)Solubility (pellet method) (%)=(1−(weight dry insoluble pelletmaterial/((weight of 20 ml of dispersion/weight of 50 ml ofdispersion)×initial weight dry protein powder)))×100  2)

The natural pH value of the protein isolate produced in Example 1 inwater (1% protein) is shown in Table 4:

TABLE 4 Natural pH of S700 solution prepared in water at 1% proteinBatch Product Natural pH S005-A 19-09A S700 3.21

The solubility results obtained are set forth in the following Tables 5and 6:

TABLE 5 Solubility of S700 at different pH values based on proteinmethod Solubility (protein method) (%) pH Nat. Batch Product pH 2 pH 3pH 4 5 pH 6 pH 7 pH S005-A19-09A S700 100 100 100 8.5 39.6 47.9 95.8

TABLE 6 Solubility of S700 at different pH values based on pellet methodSolubility (pellet method) (%) Nat. Batch Product pH 2 pH 3 pH 4 pH 5 pH6 pH 7 pH S005-A19- S700 98.7 100 98.2 20.9 45.3 64.3 98.9 09A

As can be seen from the results of Tables 5 and 6, the S700 product wasvery soluble in the pH range of 2 to 4.

Example 4

This Example contains an evaluation of the clarity in water of the soyprotein isolate produced by the method of Example 1 (S700).

The clarity of the 1% w/v protein solutions prepared as described inExample 3 was assessed by measuring the absorbance at 600 nm, with alower absorbance score indicating greater clarity. Analysis of thesamples on the HunterLab ColorQuest XE instrument in transmission modealso provided a percentage haze reading, another measure of clarity.

The clarity results are set forth in the following Tables 7 and 8:

TABLE 7 Clarity of S700 solution at different pH values as assessed byA600 A600 Batch Product pH 2 pH 3 pH 4 pH 5 pH 6 pH 7 Nat. pH S005- S7000.007 0.015 0.035 >3.0 >3.0 2.219 0.014 A19-09A

TABLE 8 Clarity of 5700 solution at different pH values as assessed byHunterLab analysis HunterLab haze reading (%) Batch Product pH 2 pH 3 pH4 pH 5 pH 6 pH 7 Nat. pH S005- S700 0.0 0.0 0.0 90.6 90.4 90.1 0.0A19-09A

As can be seen from the results of Tables 7 and 8, solutions of S700were transparent in the pH range of 2 to 4.

Example 5

This Example contains an evaluation of the solubility in a soft drink(Sprite) and sports drink (Orange Gatorade) of the soy protein isolateproduced by the method of Example 1 (S700). The solubility wasdetermined with the protein added to the beverages with no pH correctionand again with the pH of the protein fortified beverages adjusted to thelevel of the original beverages.

When the solubility was assessed with no pH correction, a sufficientamount of protein powder to supply 1 g of protein was weighed into abeaker and a small amount of beverage was added and stirred until asmooth paste formed. Additional beverage was added to bring the volumeto 50 ml, and then the solutions were stirred slowly on a magneticstirrer for 60 minutes to yield a 2% protein w/v dispersion. The proteincontent of the samples was analyzed using a LECO FP528 NitrogenDeterminator then an aliquot of the protein containing beverages wascentrifuged at 7800 g for 10 minutes and the protein content of thesupernatant measured.Solubility (%)=(% protein in supernatant/% protein in initialdispersion)×100

When the solubility was assessed with pH correction, the pH of the softdrink (Sprite) (3.39) and sports drink (Orange Gatorade) (3.19) withoutprotein was measured. A sufficient amount of protein powder to supply 1g of protein was weighed into a beaker and a small amount of beveragewas added and stirred until a smooth paste formed. Additional beveragewas added to bring the volume to approximately 45 ml, and then thesolutions were stirred slowly on a magnetic stirrer for 60 minutes. ThepH of the protein containing beverages was measured and then adjusted tothe original no-protein pH with HCl or NaOH as necessary. The totalvolume of each solution was then brought to 50 ml with additionalbeverage, yielding a 2% protein w/v dispersion. The protein content ofthe samples was analyzed using a LECO FP528 Nitrogen Determinator thenan aliquot of the protein containing beverages was centrifuged at 7800 gfor 10 minutes and the protein content of the supernatant measured.Solubility (%)=(% protein in supernatant/% protein in initialdispersion)×100

The results obtained are set forth in the following Table 9:

TABLE 9 Solubility of S700 in Sprite and Orange Gatorade no pHcorrection pH correction Solubility (%) in Solubility (%) in Solubility(%) Solubility (%) in Batch Product Sprite Orange Gatorade in SpriteOrange Gatorade S005-A19-09A S700 95.9 100 92.9 100

As can be seen from the results of Table 9, S700 was highly soluble inthe Sprite and the Orange Gatorade. As S700 is an acidified product,protein addition had little effect on beverage pH.

Example 6

This Example contains an evaluation of the clarity in a soft drink andsports drink of the soy protein isolate produced by the method ofExample 1 (S700).

The clarity of the 2% w/v protein dispersions prepared in soft drink(Sprite) and sports drink (Orange Gatorade) in Example 5 were assessedusing the methods described in Example 4. For the absorbancemeasurements at 600 nm, the spectrophotometer was blanked with theappropriate beverage before the measurement was performed.

The results obtained are set forth in the following Tables 10 and 11:

TABLE 10 Clarity (A600) of S700 in Sprite and Orange Gatorade no pHcorrection pH correction A600 in A600 in A600 in Orange A600 in OrangeBatch Product Sprite Gatorade Sprite Gatorade S005-A19-09A S700 0.0280.000 0.021 0.000

TABLE 11 Hunter Lab haze readings for S700 in Sprite and Orange Gatoradeno pH correction pH correction haze (%) in haze (%) in haze (%) in haze(%) in Batch Product Sprite Orange Gatorade Sprite Orange Gatorade noprotein 0.0 44.0 0.0 44.0 S005-A19-09A S700 1.6 51.7 0.0 41.1

As can be seen from the results of Tables 10 and 11, the S700 hadessentially no effect on the clarity of the Sprite or the OrangeGatorade.

SUMMARY OF THE DISCLOSURE

In summary of this disclosure, the present invention provides a novelprocedure for forming a soy protein product that is soluble in acidicmedia and forms heat stable and transparent solutions therein.Modifications are possible within the scope of this invention.

1. A method of producing a soy protein product with a protein content ofat least about 60 wt % (N×6.25) d.b., which comprises: (a) extracting asoy protein source with an aqueous salt solution, optionally containingan antioxidant, at a temperature of at least about 1° C. to causesolubilization of soy protein in the soy protein source and to form anaqueous protein solution having a protein content of about 5 to about 50g/L and a pH of about 1.5 to about 11, (b) separating the aqueousprotein solution from residual soy protein source, (c) optionallytreating the aqueous protein solution with an adsorbent to remove colourand/or odour compounds from the aqueous protein solution, (d) addingcalcium salt solution to the aqueous protein solution to a conductivityof about 15 to about 85 mS to cause a precipitate to form in the aqueousprotein solution, (e) removing the precipitate from the aqueous soyprotein solution, (f) increasing the protein concentration of the soyprotein solution to about 50 to about 400 g/L, while maintaining theionic strength substantially constant by using a selective membranetechnique, to provide a concentrated protein solution, (g) optionallydiafiltering the concentrated protein solution before or after completeconcentration thereof, using from about 2 to about 40 volumes ofdiafiltration solution, (h) optionally pasteurizing the concentrated andoptionally diafiltered protein solution at a temperature of about 55° toabout 70° C. for about 30 seconds to about 60 minutes, followedoptionally by cooling to a temperature of about 20° C. to about 35° C.,(i) diluting the concentrated and optionally diafiltered proteinsolution into about 2 to about 20 volumes of water having a temperatureof about 2° to about 90° C., (j) acidifying the resulting solution to apH of about 1.5 to about 4.4 to produce an acidified clear proteinsolution, (k) optionally polishing the acidified clear protein solution,(l) increasing the concentration of the acidified clear protein solutionto about 50 to about 300 g/L while maintaining the ionic strengthsubstantially constant by using a selective membrane technique toprovide a second concentrated protein solution, (m) optionallydiafiltering the second concentrated protein solution before or aftercomplete concentration thereof using from about 2 to about 40 volumes ofdiafiltration solution, (n) optionally treating the second concentratedand optionally diafiltered protein solution with an adsorbent to removecolour and/or odour compounds, and (o) optionally drying the secondconcentrated and optionally diafiltered protein solution to provide asoy protein product having a protein content of at least about 60 wt %(N×6.25) d.b.
 2. A method of producing a soy protein product with aprotein content of at least about 60 wt % (N×6.25) d.b., whichcomprises: (a) extracting a soy protein source with an aqueous saltsolution, optionally containing an antioxidant, at a temperature of atleast about 1° C. to cause solubilization of soy protein in the soyprotein source and to form an aqueous protein solution having a proteincontent of about 5 to about 50 g/L and a pH of about 1.5 to about 11,(b) separating the aqueous protein solution from residual soy proteinsource, (c) optionally treating the aqueous protein solution with anadsorbent to remove colour and/or odour compounds from the aqueousprotein solution, (d) adding calcium salt solution to the aqueousprotein solution to a conductivity of about 15 to about 85 mS to cause aprecipitate to form in the aqueous protein solution, (e) removing theprecipitate from the aqueous soy protein solution, (f) partiallyconcentrating the aqueous protein solution to a protein concentration ofabout 50 g/L or less, while maintaining the ionic strength substantiallyconstant by using a selective membrane technique, (g) optionallydiafiltering the partially concentrated protein solution, preferablyusing an aqueous salt solution of the same molarity and pH as theextraction solution, before or after partial concentration thereof usingfrom about 2 to about 40 volumes of diafiltration solution, (h) dilutingthe partially concentrated and optionally diafiltered protein solutioninto about 0.5 to about 20 volumes of water having a temperature ofabout 2° to about 90° C., (i) acidifying the resulting solution to a pHof about 1.5 to about 4.4 to produce an acidified clear proteinsolution. (j) optionally polishing the acidified clear protein solution,(k) increasing the concentration of the acidified clear protein solutionto about 50 to about 300 g/L, while maintaining the ionic strengthsubstantially constant by using a selective membrane technique toprovide a concentrated protein solution, (l) optionally diafiltering theconcentrated protein solution, preferably using water or dilute salinesolution, before or after complete concentration thereof using fromabout 2 to about 40 volumes of diafiltration solution, (m) optionallytreating the concentrated and optionally diafiltered protein solutionwith an adsorbent to remove colour and/or odour compounds, and (n)optionally pasteurizing the concentrated and optionally diafilteredprotein solution at a temperature of about 55° to about 70° C. for about30 seconds to about 60 minutes, followed optionally by cooling to atemperature of about 20° C. to about 35° C., and (o) optionally dryingthe concentrated and optionally diafiltered protein solution to providea soy protein product having a protein content of at least about 60 wt %(N×6.25) d.b.
 3. A method of producing a soy protein product with aprotein content of at least about 60 wt % (N×6.25) d.b., whichcomprises: (a) extracting a soy protein source with an aqueous saltsolution, preferably aqueous sodium chloride solution, optionallycontaining an antioxidant, at a temperature of at least about 1° C. tocause solubilization of soy protein in the soy protein source and toform an aqueous protein solution having a protein content of about 5 toabout 50 g/L and a pH of about 1.5 to about 11, (b) separating theaqueous protein solution from residual soy protein source, (c)optionally treating the aqueous protein solution with an adsorbent toremove colour and/or odour compounds from the aqueous protein solution,(d) partially concentrating the aqueous protein solution to about 50 g/Lor less while maintaining the ionic strength substantially constant byusing a selective membrane technique to provide a partially concentratedprotein solution, (e) optionally diafiltering the partially concentratedprotein solution, before or after partial concentration thereof usingfrom about 2 to about 40 volumes of diafiltration solution, (f) addingcalcium salt solution to the partially concentrated protein solution toa conductivity of about 15 to about 85 mS to cause a precipitate to formin the partially concentrated protein solution, (g) removing theprecipitate from the partially concentrated protein solution, (h)further increasing the protein concentration of the partiallyconcentrated protein solution to about 50 to about 400 g/L whilemaintaining the ionic strength substantially constant by using aselective membrane technique to provide a concentrated protein solution,(i) optionally diafiltering the concentrated protein solution,preferably using an aqueous salt solution of the same molarity and pH asthe extraction solution, before or after complete concentration thereofusing from about 2 to about 40 volumes of diafiltration solution, (j)optionally pasteurizing the concentrated and optionally diafilteredprotein solution at a temperature of about 55° to about 70° C. for about30 seconds to about 60 minutes, followed optionally by cooling to atemperature of about 20° C. to about 35° C., (k) diluting the clarifiedretentate into about 2 to about 20 volumes of water, having atemperature of about to about 90° C., (l) acidifying the resultingsolution to a pH of about 1.5 to about 4.4 to produce an acidified clearprotein solution, (m) optionally polishing the acidified clear proteinsolution, (n) increasing the concentration of the acidified clearprotein solution to a protein concentration of about 50 to about 300g/L, while maintaining the ionic strength substantially constant byusing a selective membrane technique, cut-off of about 3,000 to about1,000,000 Daltons to provide a second concentrated protein solution, (o)optionally diafiltering the second concentrated protein solution, beforeor after complete concentration thereof using from about 2 to about 40volumes of diafiltration solution, (p) optionally treating the secondconcentrated and optionally diafiltered protein solution with anadsorbent to remove colour and/or odour compounds, and (q) optionallydrying the second concentrated and optionally diafiltered proteinsolution to provide a soy protein product having a protein content of atleast about 60 wt % (N×6.25) d.b.
 4. The method of any one of claims 1,2 and 3 wherein at least one of said concentration steps and/or at leastone of the optional diafiltration steps are operated in a mannerfavourable to the removal of trypsin inhibitors.
 5. The method of anyone of claims 1, 2 and 3 wherein said concentration and optionaldiafiltration steps are carried out at a temperature of about 2 to about60° C.
 6. The method of any one of claims 1, 2 and 3 wherein a reducingagent is present or added to disrupt or rearrange the disulfide bonds oftrypsin inhibitors to achieve a reduction in trypsin inhibitor activityduring the extraction step and/or during the concentration steps andoptional diafiltration steps and/or prior to drying and/or the dried soyprotein product.
 7. The method of any one of claims 1, 2 and 3 whereinsaid acidified soy protein solution, said partially concentrated soyprotein solution and/or said concentrated soy protein solution, issubjected to a heat treatment, to inactivate heat labileanti-nutritional factors, including heat-labile trypsin inhibitors, saidheat treatment being effected at a temperature of about 70° to about160° C. for about 10 seconds to about 60 minutes, the heat treated soyprotein solution optionally thereafter being cooled to a temperature ofabout 2° to about 60° C. for further processing.
 8. The method of anyone of claims 1, 2 and 3 wherein the soy protein product has a proteincontent of at least about 90 wt %.
 9. The method of any one of claims 1,2 and 3 wherein the soy protein product has a protein content of atleast about 100 wt %.
 10. The method of any one of claims 1, 2 and 3wherein said aqueous salt solution is sodium chloride solution.
 11. Themethod of any one of claims 1, 2 and 3 wherein said extraction step iseffected at a temperature of about 15° to about 35° C. to form anaqueous solution having a protein content of about 10 to about 50 g/Land a pH of about 5 to about
 7. 12. The method of claim 1 or 2 whereinthe calcium salt solution is calcium chloride solution and is added tothe aqueous protein solution to a conductivity of about 17 to about 25mS.
 13. The method of claim 1 wherein the protein concentration of thesoy protein solution is increased to about 100 to about 250 g/Lemploying a membrane having a molecular weight cut-off of about 3,000 toabout 1,000,000 Daltons.
 14. The method of claim 13 wherein themolecular weight cut-off is about 5,000 to about 100,000 Daltons. 15.The method of claim 1 wherein the concentrated protein solution isdiafiltered using a salt solution of the same molarity and pH of theextraction solution using from about 2 to about 40 volumes ofdiafiltration solution using a membrane having a molecular weightcut-off of about 3,000 to about 1,000,000 Daltons.
 16. The method ofclaim 15 wherein about 5 to about 25 volumes of diafiltration solutionis used with an antioxidant present during at least part of thediafiltration step and the membrane has a molecular weight cut-off ofabout 5,000 to about 100,000 Daltons.
 17. The method of claim 15 whereinsaid diafiltration step is effected until no significant furtherquantities of contaminants or visible colour are present in thepermeate.
 18. The method of claim 1 wherein said pasteurization step iseffected at a temperature of about 60° to about 65° C. for about 10 toabout 15 minutes.
 19. The method of claim 1 wherein the concentrated andoptionally diafiltered protein solution is diluted into about 10 toabout 15 volumes of water having a temperature of about 10° to about 50°C.
 20. The method of claim 19 wherein the temperature is about 20° toabout 30° C.
 21. The method of claim 1 wherein the acidifying step iseffected to a pH of about 2.0 to about 4.0.
 22. The method of claim 1wherein the protein concentration of the acidified clear proteinsolution is increased to about 100 to about 200 g/L using a membranehaving a molecular weight cut-off of about 3,000 to about 1,000,000Daltons.
 23. The method of claim 22 wherein the molecular weight cut-offis about 5,000 to about 100,000 Daltons.
 24. The method of claim 1wherein the second concentrated protein solution is diafiltered usingwater or dilute saline solution using from about 2 to about 40 volumesof diafiltration solution using a membrane having a molecular weightcut-off of about 3,000 to about 1,000,000 Daltons.
 25. The method ofclaim 24 wherein about 5 to about 25 volumes of diafiltration solutionare used with an antioxidant present during at least part of thediafiltration step and the membrane has a molecular weight cut-off ofabout 5,000 to about 100,000 Daltons.
 26. The method of claim 25 whereinthe diafiltered is effected until no significant further quantities ofcontaminants or visible colour are present in the permeate.
 27. Themethod of claim 2 or 3 wherein the partially concentrated proteinsolution is effected using a membrane having a molecular weight cut-offof about 3,000 to about 1,000,000 Daltons.
 28. The method of claim 27wherein the membrane has a molecular weight cut-off of about 5,000 toabout 100,000 Daltons.
 29. The method of claim 2 or 3 wherein thepartially concentrated protein solution is diafiltered using an aqueoussalt solution of the same molarity and pH as extraction solution usingabout 5 to about 25 volumes of diafiltration solution using a membranehaving a molecular weight cut-off of about 3,000 to about 1,000,000Daltons.
 30. The method of claim 29 wherein said diafiltration iseffected with an antioxidant present during at least part of thediafiltration step and the membrane by a molecular weight cut-off ofabout 5,000 to about 100,000 Daltons.
 31. The method of claim 30 whereinsaid diafiltered is effected until no significant further quantities ofcontaminants or visible colour are present in the permeate.
 32. Themethod of claim 2 wherein the partially concentrated and optionallydiafiltered protein solution is diluted into about 1 to about 10 volumesof water having a temperature of about 10° to about 50° C.
 33. Themethod of claim 32 wherein said dilution is effected using about 2 toabout 5 volumes of water having a temperature of about 20° to about 30°C.
 34. The method of claim 2 wherein the acidifying step is effected toa pH of about 2.0 to about 4.0.
 35. The method of claim 2 wherein theacidified clear protein solution is concentrated to about 100 to about200 g/L using a membrane having a molecular weight cut-off of about3,000 to about 1,000,000 Daltons.
 36. The method of claim 35 wherein themembrane has a molecular weight cut-off of about 5,000 to about 100,000Daltons.
 37. The method of claim 2 or 3 wherein the concentrated proteinsolution is diafiltered using water or dilute saline solution having 5to about 25 volumes of diafiltration solution using a membrane having amolecular weight cut-off of about 3,000 to about 1,000,000 Daltons. 38.The method of claim 37 wherein the diafiltration is effected with anantioxidant present during at least part of the diafiltration step usinga membrane having a molecular weight cut-off of about 5,000 to about100,000 Daltons.
 39. The method of claim 38 wherein the diafiltration iseffected until no significant further quantities of contaminants orvisible colour are present in the permeate.
 40. The method of claim 2 or3 wherein the concentrated and optionally diafiltered solution ispasteurized at a temperature of about 60° to about 65° C. for about 10to about 15 minutes.
 41. The method of claim 3, wherein the calcium saltsolution is calcium chloride and is added to partially concentratedprotein solution to a conductivity of about 17 to about 25 mS.
 42. Themethod of claim 3 wherein the concentration of the partiallyconcentrated protein solution is further increased to about 100 to about250 g/L.
 43. The method of claim 42 wherein the further concentration iseffected using a membrane having a molecular weight cut-off of about3,000 to about 1,000,000 Daltons.
 44. The method of claim 43 wherein themolecular weight cut-off is about 5,000 to about 100,000 Daltons. 45.The method of claim 3 wherein the clarified retentate is diluted intoabout 10 to about 15 volumes of water having a temperature of about 10°to about 50° C.
 46. The method of claim 45 wherein the temperature isabout 20° to about 30° C.
 47. The method of claim 3 wherein theacidification step is to a pH of about 2 to about 4.0.
 48. The method ofclaim 3 wherein the acidified clear protein solution is concentrated toa concentration of about 100 to about 200 g/L.
 49. The method of claim48 wherein said selective membrane technique is effected using amembrane having a molecular weight cut-off of about 3,000 to about1,000,000 Daltons.
 50. The method of claim 49 wherein the molecularweight cut-off is about 5,000 to about 100,000 Daltons.
 51. The methodof claim 3 wherein the second concentrated protein solution isdiafiltered using water or dilute saline solution using about 5 to about25 volumes of diafiltration solution using a membrane with a molecularweight cut-off of about 3,000 to about 1,000,000 Daltons.
 52. The methodof claim 51 wherein said diafiltration is effected with an antioxidantpresent during at least part of the diafiltration step using a membranewith a molecular weight cut-off of about 5,000 to about 100,000 Daltons.53. The method of claim 52 wherein said diafiltration is effected untilno significant further quantities of contaminants or visible colour arepresent in the permeate.
 54. The method of claim 5 wherein saidtemperature is about 20° to about 25° C.
 55. The method of claim 7wherein said heat treatment is effected at about 80° to about 120° C. toabout 30 seconds to about 5 minutes.
 56. The method of claim 7 whereinsaid heat treatment is effected at about 85° to about 95° C. for about30 seconds to about 5 minutes.
 57. The method of claim 55 wherein theheat treated soy protein solution is cooled to about 20° to about 35° C.